1. Field of the Invention
The present invention relates generally to modification of adenoviral gene therapy vectors and uses thereof. In one embodiment, the present invention discloses modified adenoviral vectors that have altered tropism. In another embodiment, there is provided fluorescently labeled adenoviral vectors useful for monitoring viral vector distribution.
2. Description of the Related Art
Adenoviral vectors (Ad) have proven to be of enormous utility for a variety of gene therapy applications. This usefulness is derived largely from the unparalleled delivery efficiency of these vectors for in vitro and in vivo applications. Despite this property, however, the full benefit of these vectors is undermined currently by the lack of cell-specific gene delivery capability. Specifically, the promiscuous tropism of the adenovirus hinders gene delivery in a targeted, cell-specific manner. Thus, for the many gene therapy applications where such cell-specific transduction is required, current adenoviral vectors have limited utility.
To address the issue of efficient, cell-specific gene delivery, a variety of strategies have been developed to alter adenoviral tropism. These approaches have included direct chemical modifications of the adenoviral capsid proteins, bi-specific complexes (i.e., a capsid protein and a targeting moiety), and genetic capsid modifications (i.e., genetic replacement/insertion). Whereas the former two strategies have established the feasibility of adenoviral re-targeting, practical production issues as well as regulatory approval considerations have placed the utmost importance on the approach in which modifications to the adenoviral tropism are introduced genetically.
To this end, methods that alter adenoviral tropism via modifications of the adenoviral major capsid proteins, fiber, penton and hexon have expanded tropism such that it is independent of the native adenoviral receptor (CAR). These methods additionally ablate the native tropism of the adenovirus. Experimentally, tropism expansion has been achieved via the incorporation of peptide ligands with specificity for target cellular markers. This has largely been via the incorporation of the peptide, RGD-4C, at fiber and hexon locales. RGD-4C recognizes integrins of the αvβ3 and αvβ5 classes. In addition, other small peptide markers have been employed to the same end. These studies have established that genetic modification(s) to the capsid can indeed alter adenoviral vector tropism to achieve a limited and/or specific range of gene delivery.
Of note, the locales employed in the context of modifying the major capsid proteins for targeting purposes have allowed only the incorporation of small peptides. To date, these have consisted of peptides identified via phage display methods, or short physiologic peptide ligands. Both of these types of targeting motifs, however, are suboptimal with respect to accomplishing the goal of cell-specific delivery. With respect to the former, only an extremely limited repertoire of useful peptides have been identified heretofore via phage display techniques. In addition, these peptides have tended to be of low affinity. Furthermore, the fidelity of such targeting peptides, when in the context of the adenoviral vector, is not always preserved. With respect to the latter, available physiologic peptides do not allow targeting to the range of cells required for practical gene therapy approaches.
In this regard, single chain antibodies (scFvs) represent motifs with highly diverse specificities that can be exploited for adenoviral targeting. In addition, single chain antibodies possess high affinities for cognate targets. On this basis, the ability to incorporate single chain antibodies into the adenoviral capsid, and for the single chain antibody specificity/affinity to be preserved following display of the chimeric/recombinant capsid protein would dramatically enhance the utility of genetic capsid modification methods for adenoviral retargeting. The inability to configure single chain antibodies at fiber, hexon, and penton locales has indicated the need to examine the ability of single chain antibodies to be incorporated into alternate capsid proteins.
Thus, the prior art is deficient in modification of adenoviral vectors that allows for genetic introduction of a useful targeting moiety. The present invention fulfills this long-standing need and desire in the art.